1. Field of the Invention
This invention relates generally to a method of isolating a normal mammalian bronchial or bronchiolar epithelial lung cell; to the isolated mammalian epithelial lung cells produced by the method; to the use of the isolated mammalian epithelial lung cell for the production of proteins and as an assay system for pituitary factors which promote the growth of such mammalian epithelial lung cells. The unique method of isolating the lung cells results in novel bronchial or bronchiolar epithelial cells isolated from mammalian lung which grow in serum-free defined medium and which exhibit accelerated growth in the presence of mammalian pituitary extract.
2. Background and Prior Art
The lung is a complex organ composed of over 40 different cell types. Work on small cell carcinoma and recent advances in endocrinology have led to the recognition that the lung is the site of production of, and target tissue for a number of endocrine, paracrine and autocrine factors. While several tissue culture systems have been reported for primary culture of cells from the lung, specifically tracheobronchial epithelium (5,15,16,27,31), epithelial cell lines which can maintain their differentiated function in vitro have been difficult to establish without viral transformation or immortalization by transfection with various oncogenes. Reddel et al., PCT 89/03994 discloses human bronchial epithelial cells after viral transformation capable of growth in culture. These cells were transformed with SV40 or adenovirus-12 SV40 hybrid virus or with a recombinant plasmid containing portions of the Rous sarcoma virus. Clearly these cells are not the same as the normal cells of the present invention which do not contain such a transforming virus.
Serum is known to support the growth of many cell types, however it is complex and not well defined. In vivo, a cell would be exposed to the equivalent of serum only under special circumstances involving tissue injury and blood coagulation. In vitro, serum may not support the growth of some cell types, due to specific inhibition or a failure to provide an adequate concentration of stimulatory factors. For this reason we sought to utilize hormone-supplemented. serum-free medium as a method of selecting for specific cell types from the lung. Mather and Sato demonstrated that a serum-free hormonally defined medium for melanoma cells could be used to select for that same cell type when used as the culture media for a mixed cell population (23). Piltch et al were able to select for differentiated epithelial cells from rat thymus, and maintain these cells continuously in a defined serum-free medium supplemented with hormones (25). Loo and co-workers have described a serum-free, hormonally defined culture system for the establishment of a mouse embryo cell, selected from whole embryos. These cultures, when carried in the presence of serum, undergo a well-defined crisis or senescence (17). This senescence does not occur when these cultures are carried continuously in serum-free, hormonally defined culture. The inventors believe that non-transformed epithelial cell lines from normal tissue would be of great use in furthering our understanding of lung endocrinology and physiology. The inventors also believe that such cells would find use in the production of proteins and as an assay cell line responding to growth promoting factors thereby enabling the isolation of such growth promoting factors.